Epidermal growth factor (EGF) and its receptor (EGF-R) have a fundamental role in controlling cell growth, differentiation and neoplasia. The response of cells to EGF clearly involves multiple messengers (or "signal transducers") to conduct the initial signal at the membrane to the nuclear transcription machinery, resulting in regulation of specific gene expression. Although several signal transduction messengers and pathways are known to be altered by EGF treatment of cells, the precise functional role of these putative transducers and the specific which must transpire in the process of EGF regulation of gene transcription remain unclear. We have established a transient co-transfection model system in highly differentiated cultured GH4 rat pituitary cells whereby an EGF responsive prolactin promoter-luciferase construct (prPRLluc) provides a functional assay for the effects of EGF on lactotroph specific gene expression. This system also provides a model for EGF induced differentiation, in contrast to the mitogenic effects of EGF on other cell lines. In previous studies, I have discovered that EGF-mediated regulation of the rPRL promoter is Ras-independent and that EGF and Ras employ mutually antagonistic signal transduction pathways to regulate the prolactin gene promoter. Neither Raf and MAP kinase, nor Ets-1/GHF1 are required for the EGF response. Furthermore, the EGF response maps to the FP II region of the rPRL promoter, a region known to bind a repressor (F2F) of rPRL gene expression. These findings are novel and are likely to be important to our understanding of the mechanisms by which a ubiquitous growth factor/growth factor receptor is able to produce specific effects on cells of different function. The overall goal of this grant proposal is to define the mediators and pathway whereby EGF regulates PRL gene transcription in the GH4 pituitary cell. To achieve these goals, I propose four specific aims. First, I plan to precisely map the EGF response element using site-specific mutations and heterologous promoters. I also plan to identify and characterize the trans-activating factor responsible for EGF activation of the rPRL promoter, and examine the relationship of this factor to the previously identified F2F utilizing gel-shift assays, UV-crosslinking, Southwestern analysis. In addition, the role of protein kinase C (PKC) in mediating the EGF effect will be extensively explored. This will require the use of specific PKC inhibitors, the identification of specific EGF-induced PKC isozyme translocation, and subsequently the overexpression of these specific isozymes in the GH4 cell. Finally, I plan to explore the role of PLCgamma-1 in the EGF response. This will involve examining the effect of expression of both PLCgamma-1 inhibitory and wild-type constructs on the EGF response. In addition, PCR amplified and cloned SH2/SH3 domains of PLCgamma-1 will be utilized as inhibitors of the PLCgamma-1 component of the EGF signaling pathway. I will also employ immunoprecipitation and Western blotting to document EGF-induced recruitment of PLCgamma-1 to the EGF-R, and phosphorylation of PLCgamma-1. These studies should provide important insights into the mechanism of EGF-dependent gene regulation.